This proposal examines a novel protein kinase called PKK which associates with protein kinase C beta which can activate NF kappa B, and which is required for the activation of NF kappa B by phorbol ester. PKK is also expressed at very high levels in some human breast cancer cell lines. One aim is to understand how PKK is activated in terms of ligands, receptors, and upstream activators. PKK is heavily phosphorylated and the role of PKK phosphorylation in catalytic activation and the ability of PKK to activate NFkappa B will be explored in detail. Since certain MAP3 Kinases interact with PKK, a possible role of these MAP3Ks as upstream activators of PKK will be explored. The identity of putative upstream activating kinases that may participate in the phosphorylation of a critical activation loop residue of PKK will also be sought. The identification of upstream activators will narrow the search for possible ligands and pathways involved in PKK activation in a number of cell types. Specific dominant negative forms of PKK and the use of stable siRNA constructs will facilitate these studies. A second aim is to understand how PKK activates NF kappa B. The structural features of PKK that contribute to NFkappa B activation will be examined in detail using site-directed mutagenesis. Our studies will explore how PKK interacts directly or indirectly with components of the IKK complex. Finally we will explore whether PKK contributes to NFkappa B activation in breast cancer and whether PKK is required for tumorigenesis. The potential role of PKK in mammary epithelial differentiation will be explored and PKK's role in mammary development will be analyzed using a conditional knockout approach. PKK represents a novel enzyme that may be critical in pathways that are important in cancer, inflammation, cell survival, and differentiation.